PROJECT SUMMARY Effective treatments are elusive for the majority of patients with neuropathic pain. This proposal will validate nuclear factor erythroid 2-related factor 2 (Nrf2)?a transcription factor?as a new therapeutic target for neuropathic pain. Nrf2 induces >200 antioxidant-related genes and can be activated with a single drug. Because reactive oxygen and nitrogen species (ROS/RNS) are a key node for neuropathic pain?driving mitochondrial dysfunction, cytokine production, and neuronal hyperexcitability?stimulation of endogenous antioxidants is predicted to simultaneously resolve multiple neuropathic pain mechanisms. However, the full extent to which Nrf2 activity regulates neuropathic pain behaviors and underlying cellular mechanisms is not known, hindering the realization of Nrf2 as new drug target. The overall objective of this application is to validate Nrf2 activation as a disease-modifying therapeutic target for neuropathic pain with no abuse liability. Our central hypothesis is that Nrf2 activation increases multiple antioxidants, therefore reversing neuropathic pain behaviors and counteracting neuropathic pain mechanisms that are driven by ROS/RNS, without inducing reward. The basis for this hypothesis is our finding that a pharmacological Nrf2 activator can alleviate such behavioral and biochemical correlates of neuropathic pain. The rationale is that by testing this hypothesis in vivo in rodents, and in vitro in human and mouse tissue, we expect to translationally validate the Nrf2 pathway as a new, non-addictive, and disease-modifying therapeutic target. To accomplish the overall objective of this application, we will test our central hypothesis by modulating the activity of Nrf2 using pharmacologic and genetic methods. The chronic constriction injury and methylglyoxal-induced neuropathy models of peripheral neuropathic pain will be used, and behavioral and mechanistic endpoints will be assessed using reflex and operant assays, and a range of molecular biology, microscopy, and live-cell imaging techniques. These approaches will be employed across the following specific aims: 1) Validate Nrf2 as a regulator of neuropathic pain behaviors and mechanisms in vivo; 2) Validate Nrf2 as a regulator of the pronociceptive functions of human DRG neurons and microglia; and 3) Determine the abuse liability of Nrf2 activation in vivo. Our proposal is highly innovative, in our opinion, because it targets an uninvestigated regulatory pathway to alleviate neuropathic pain. The results will have a significant impact by translationally validating the Nrf2 pathway as a new, non-addictive, and disease-modifying therapeutic target for neuropathic pain.